2.3. FTIR Spectra of ICFs and PSCs
Considering the strong infrared absorption of H
2O at the amide I region, we used D
2O as the dispersion solution in FTIR analysis [
13].
Figure 2A gives second derivative spectra and Fourier self-deconvolution (FSD) spectra for ICFs at 5 °C and 50 °C and the same set of spectra are given for PSCs in
Figure 2B. The spectral changes detected by the FSD analyses were in agreement with those observed by the second derivative analysis. Prystupa and Donald [
16] reported that the spectra obtained from D
2O solutions were of better quality than those obtained for H
2O solutions.
Figure 3 shows the amide I region in the FSD spectra of ICFs and PSCs dispersed in D
2O (5%
w/
v) at 5 °C. The spectra were resolved to the underlying bands by a combination of FSD spectra and peak-fitting. The well-established empirical structure-frequency correlations (
Table 2) indicate that β-sheet has a strong absorption band near 1631 cm
−1 and a weaker band at high frequency (>1680 cm
−1), whereas β-turn is reported at frequency near 1663–1671 cm
−1. The peaks for α-helix and 3
10-helix are located at ~1653 and ~1641 cm
−1, respectively. Random coil structure is generally assigned to the band around 1645 cm
−1.
In the present study, the amide I band absorption spectral profiles of ICFs and PSCs are evidently different. Clearly, at 5 °C, the ICFs spectrum exhibits five components (
Figure 3A) while PSCs spectrum exhibits seven components (
Figure 3B). The multiple structures of the collagen amide I are due to the heterogeneity of its peptide C=O group in a triple helix, a factor that directly influences the profile of the amide I band FTIR spectrum [
17]. The major component of ICFs appears at ~1657 cm
−1 (65.4% area), which means ICFs has predominant α-helical structure. In comparison, PSCs contain more β-turn (~1665 cm
−1, 30.3%) and random coil (~1647 cm
−1, 17.8%) than α-helix (~1655 cm
−1, 16.1%). Collagen triple helices naturally aggregate to form fibrils and fibre bundles, which are stabilized by inter- and intramolecular cross-links, resulting in insoluble structures with higher molecular weight and thermostability [
18,
19,
20].
2.4. FTIR Spectra as a Function of Temperature during Heating and Cooling of ICFs or PSCs
The changes in the Fourier self-deconvolved infrared spectra in the amide I region upon heating (from 5 °C to 70 °C) and cooling (from 70 °C to 5 °C) of ICFs or PSCs dispersed in D
2O (5%
w/
v) are shown in
Figure 4.
As shown in
Figure 4A, ICFs remained unchanged up to 40 °C, but were tremendously modified above 45 °C, corresponding to the transition temperature obtained by DSC (
Figure 1). More marked changes in ICFs took place since 50 °C, which seemed a complete loss of α-helical structure (~1657 cm
−1) relative to the α-helix content at 5 °C. In parallel, the bands at ~1631 and ~1682 cm
−1 gradually decrease during the heating, but they change in the opposite way during the cooling (
Figure 4B), as a slight increase occurs at ~1682, ~1657 and ~1631 cm
−1.
The spectrum of PSCs shows no obvious differences until up to 35 °C (
Figure 4C). At 35 °C, a new shoulder appears at ~1673 cm
−1. Meanwhile, the bands at ~1647, ~1638 and ~1629 cm
−1 all gradually increase. The total intensity of the amide I band begins to decrease at 45 °C, while the shoulder at ~1673 cm
−1 vanishes gradually and the spectral shape becomes flat. During the cooling, no clear changes are revealed until down to 30 °C, while the peaks around ~1681, ~1669, ~1660, ~1651, ~1643, ~1632 cm
−1 change subtly (
Figure 4D).
The peaks both in the spectra of ICFs and PSCs at around 1656 cm
−1 are characteristic of the amide I band between 1700 and 1600 cm
−1. It was suggested that accurate prediction of the amide I band could be contoured from the three-dimensional structure of a protein [
21]. ICFs and PSCs from the sea cucumber belong to type I collagen [
3,
9], which is constituted dominantly by the heterotrimers of two α
1 (I) and one α
2 (I) chains [
7]. Bryan et al. [
22] studied the thermal transition of a collagen model peptide by FTIR, they find that during denaturation, the band at 1645 cm
−1 significantly decreased in intensity as temperature is increased, while the 1629 cm-1 band showed small changes, and the high wavenumber band (1667 cm
−1) appears to broaden and disappear into the underlying broad feature. Our results showed that the intensity of the amide I band diminished and rebounded in the heating and cooling processes. These results revealed that the liberations might be responsible for weakening, breaking, re-forming and strengthening of the hydrogen bonds [
23]. The results here are really based on the hypothesis that the amide I excitation energy strongly depends on the length and orientation of associated hydrogen bonds [
24].
2.5. 2D Correlation Analysis of ICFs and PSCs
The one-dimensional analysis was further extended to get more information on the sequence of structural events by 2D COS.
Figure 5 displays 2D synchronous and asynchronous correlation maps of whole ICFs constructed from temperature-dependent spectral variations of heating process (from 5 to 70 °C) in the amide I region (1700–1600 cm
−1). The synchronous correlation map (
Figure 5A) shows a prominent auto-peak (at the diagonal) at 1656 cm
−1 and two small cross-peaks (off the diagonal) at 1656/1631 and 1683/1656 cm
−1. The asynchronous correlation map (
Figure 5B) shows a positive cross-peak at 1683/1656 cm
−1 and a negative one at 1656/1631 cm
−1. A combined analysis of the signs in both synchronous and asynchronous maps in
Table 3 reveals that the decrease in proportion of α-helix structures (1656 cm
−1) happens after the decline in the proportions of β-sheet and β-turn structures (1631 and 1683 cm
−1) in ICFs during the heating.
Figure 6 illustrates synchronous and asynchronous correlation maps of ICFs during the cooling from 70 to 5 °C. The synchronous map (
Figure 6A) reveals four evident auto-peaks at 1634, 1646, 1657 and 1683 cm
−1, respectively. The positive cross-peaks at 1683/1657, 1683/1646, 1683/1634, 1657/1646, 1657/1634 and 1646/1634 indicate that all these bands increase with temperature reduction. These results are well consistent with the 1D IR spectrum of
Figure 4B. In addition, there are some negative cross-peaks (1634, 1646, 1657 and 1683 vs. 1695 cm
−1). The asynchronous map (
Figure 6B) shows positive cross-peaks at 1695 vs. 1611, 1634, 1657 and 1683 cm
−1, and negative peaks at 1683 vs. 1611, 1634, 1646 and 1657 cm
−1; 1657 vs. 1611 cm
−1; 1646 vs. 1611 cm
−1; and 1634 vs. 1611 cm
−1. The sequences of band intensity changes based on the signs of the cross-peaks in the synchronous and asynchronous maps are summarized in
Table 4. ICFs experienced the following sequence of spectral changes during cooling: (1) increase in proportion of β-sheet structure (1634 cm
−1); (2) increase in proportion of random coil structure (1646 cm
−1); (3) increase in proportion of α-helix structure (1657 cm
−1); (4) increase in proportion of β-turn structure (1683 cm
−1); (5) decrease in proportion of β-turn structure (1695 cm
−1). This sequence is contrary to the result in the heating process.
Figure 7 shows synchronous and asynchronous correlation spectra of PSCs during heating. The band at 1621 cm
−1 assigned to β-sheet structure was not considered owing to its fluctuation with temperature rise, which complicated the interpretation of the 2D COS results. The synchronous map (
Figure 7A) shows two strong auto-peaks (1656 and 1664 cm
−1) and five weak auto-peaks (1630, 1637, 1648, 1673 and 1686 cm
−1). Positive cross-peaks appear at 1637, 1648, 1656, 1664 vs. 1630 cm
−1; 1648, 1656, 1664, 1673 vs. 1637 cm
−1; 1656, 1664, 1686 vs. 1648 cm
−1; 1664, 1686 vs. 1656 cm
−1; 1686 vs. 1664 cm
−1 while there is no negative cross-peak. The asynchronous map (
Figure 7B) shows positive cross-peaks at 1673 vs. 1637, 1648, 1656, 1664 cm
−1 and negative cross-peaks at 1686 vs. 1637, 1648, 1656, 1664, 1673 cm
−1; 1664 vs. 1637 cm
−1; 1656 vs. 1637 cm
−1; 1648 vs. 1637 cm
−1. The sequences of band intensity changes based on the signs of the cross-peaks in the synchronous and asynchronous maps are summarized in
Table 5. PSCs experienced the following sequence of spectral changes during the heating: (1) decrease in proportion of β-turn structure (1673 cm
−1); (2) decrease in proportion of β-sheet structure (1637 and 1630 cm
−1); (3) decrease in proportions of random coil structure (1648 cm
−1), α-helix structure (1656 cm
−1) and β-turn structure (1664 cm
−1); (4) decrease in proportion of β-turn structure (1686 cm
−1).
Figure 8 displays synchronous and asynchronous correlation spectra of PSCs during cooling. The synchronous map shows three strong auto-peaks (1639, 1647 and 1656 cm
−1) and four weak auto-peaks (1629, 1664, 1675 and 1689 cm
−1) (
Figure 8A). The sequences of band intensity changes based on the signs of the cross-peaks in the synchronous and asynchronous correlation maps are summarized in
Table 6.
A combined analysis of the signs in both synchronous and asynchronous maps [
25] reveals the following sequence of events during cooling: (1) increase in proportion of β-turn structure (1664 cm
−1); (2) increase in proportion of α-helix structure (1656 cm
−1); (3) increase in proportions of β-turn structure (1629 cm
−1), 3
10-helix structure (1639 cm
−1), β-sheet structure (1675 cm
−1) and random coil structure (1647 cm
−1); (4) increase in proportion of β-sheet structure (1618 cm
−1); (5) decrease in proportion of β-turn structure (1689 cm
−1).
As a whole, the α-helix and β-sheet structures of both ICFs and PSCs are quite sensitive to temperature changes. The α-helix is driven especially by the formation of hydrogen bond network along the polypeptide backbone [
26]. The β-sheet structure was derived from the α-helical structures [
27]. Our study shows that along with the temperature rise, the proportion of α-helix diminishes with the increasing proportion of β-sheet structure, and the changes of β-sheet always occur before those of α-helix. These results agree well with previous research [
28,
29], indicating the collagen-unfold structures were formed during the heating process. In a collagen-unfold structure, the aligned α-helical domain is divided into single chains, which suggests that the regenerative β-sheet is the intermediate instead of single chains and is very unstable [
16,
28]. As a consequence, the partial β-sheet bands in ICFs disappeared during the cooling, which indicate a partial reverse of the collagen-fold structures [
16]. However, ICFs and PSCs showed some differences. The denaturation temperature of ICFs (45.1 °C) was 10.8 °C higher than that of PSCs, which was an earlier disappearing signal of the triple helical structure in the event sequence of PSCs. In addition, ICFs had a partial refolding ability during the cooling, while was not observed in PSCs. Inter and intramolecular cross-links in ICFs may have an important effect for the thermal stability and the refolding ability during the cooling process.